In industries producing vaccines by means of cell culture or producing biological substances secreted either by adherent or suspension cells or by microorganisms, use is made of microcarriers, roller bottles, Roux flasks, multiple chambered systems, and fermentors.
Microcarriers are small microscopic beads on which adherent cells are caused to grow. The beads are maintained in suspension in a culture medium, hereafter called the liquid phase, and with the help of a stirring system, the liquid phase is moved in contact with a gas phase. Everything is contained in a tank called a fermentor. This technique frequently used in the vaccine industry is nonetheless confronted with two major difficulties: (i) carrying out a suspension of microcarriers in a culture medium which is compatible with cell anchorage and growth conditions; and (ii) ensuring the control and stability of the culture medium pH, taking into account the fact that the exchange surface between the gas phase and the liquid phase is substantially limited.
Fermentors are also used for the culture of suspension cells or microorganisms. The handling difficulties are similar to those encountered during cultivation with a microcarrier. As the exchange surface between the gas phase and the liquid phase is limited by the fermentor diameter, the pH stability becomes extremely difficult to ensure.
Roller bottles are generally of cylindrical shape and are designed to be rotatable about their axes. The interior surface of the bottle is intended for cultivating adherent cells thereby forming a culture surface. The liquid culture medium is introduced into the bottle together with the cells. Rotation of the bottle allows the culture surface to be covered with a film of the culture medium thereby allowing cell growth on the culture surface. The culture surface is therefore limited to the size of the bottle. If the production of a large quantity of cells is desired, a large number of bottles will be necessary. This is the case for industries producing, for example, interferon, insulin, viral vaccines, or lymphokines. The handling of numerous bottles during inoculation, medium change, virus introduction, supernatant, and cell harvesting increases the risk of bottle and content contamination and requires the use of a significant number of staff. Many apparatuses have been developed to increase the culture surface of roller bottles by increasing the surface of the bottle itself. These known apparatuses are intended for developing adherent cell culture but are not intended for providing a homogeneous suspension of microcarriers or of suspension cells.
Roux flasks have been used for more than a century for producing viruses, as well as other biological substances. They have the same drawbacks as roller bottles, that is to say a limited culture surface per bottle and require a significant number of staff for handling.
In European Patent Application 0,345,415, a bottle apparatus is disclosed having an enlarged culture surface obtained by providing the bottle body with corrugations which extend axially or longitudinally relative to the bottle axis. This bottle does not, however, allow liquid stirring, and therefore it does not permit cultivation with microcarriers.
The surface used for cell development is also increased in an apparatus disclosed in U.S. Pat. No. 3,839,155 of McAleer et al. through a parallel arrangement of discs closely spaced along the bottle axis. This apparatus however is not entirely satisfactory for adherent cells because the trays, when rotated through a horizontal position, cannot retain a volume of liquid containing the cell suspension. This causes the liquid to quickly run out from the discs which does not favor cell attachment during cultivation. Moreover, this apparatus is not appropriate for the cultivation of so-called non-adherent cells because nothing is provided therein for stirring of the liquid phase.
In Luxembourg Patent Application 51,646 of Kamphans, a cell culture apparatus is shown comprising a set of parallel trays placed one above the other so as to form culture chambers. The trays are placed inside a housing which can be turned around an axis. This system permits cultivation of adherent cells on one side of the trays only. The interior surface is not used as a culture surface. Moreover, the housing does not permit the introduction of a stirring system for allowing microcarriers or microorganisms to be maintained in a homogeneous and continuous suspension.
In U.S. Pat. No. 3,925,165 of Muller, another apparatus is disclosed provided with radially-arranged vanes extending longitudinally inside a container which are continuously rotated through a gas phase at an upper part of the container and a reaction liquid in the lower part of the container. However, the vanes in this apparatus do not retain reaction fluid for an extended interval of time and therefore are not suitable for use in effectively culturing adherent cells.